Bone graft material for osseous defects and method of making same

ABSTRACT

Complexes of reconstituted collagen and demineralized bone particles or reconstituted collagen and a solubilized bone morphogenetic protein fabricated in a sponge suitable for in vivo implantation in osseous defects are disclosed. Both demineralized bone particles (DBP) and bone morphogenetic protein have demonstrated the ability to induce the formation of osseous tissue in animal and human experiments. Reconstituted collagen conjugate is highly biocapatible and can be fabricated in a variety of configurations, especially as a sponge. This material can be used as a grafting implant in plastic and reconstructive surgery, periodontal bone grafting, and in endodontic procedures. Structural durability is enhanced by cross-linking with glutaraldehyde which is also used to sterilize and disinfect the collagen conjugate prior to implantation.

BACKGROUND OF THE INVENTION

This invention relates to a biocompatible, osteogenic, collagenconjugate material, particularly in the form of a sponge, and to aprocess for making this material. While individual components of thissystem are known in the art and in the related literature, describedherein is a novel composite material, specifically designed to induceosteogenesis within its porous structure, completely biocompatible andnon-inflammatory, and ultimately resorbed and replaced by calcified,hard tissue.

Collagen was chosen as the binding matrix and main structural componentof the novel graft material herein described for several reasons:

(1) Reconstituted collagen has demonstrated excellent histocapatibilitywithout antibody formation or graft rejection in numerous in vivoimplantation studies.

(2) Reconstituted collagen can be fabricated into porous sponge-likestructures which allow unimpeded cellular ingrowth.

(3) Collagen is the natural biomaterial which constitutes from 50 to 70%by weight of the bone organic matrix.

(4) Reconstituted collagen has demonstrated the ability to bind bothlarge and small molecular weight macromolecules and complexation withcollagen protects these macromolecules from denaturation due toenvironmental influences, such as glutaraldehyde cross-linking or theeffect of other chemical agents; see M. Chvapil et al, InternationalReview of Connective Tissue Research, Vol. 6, (1973), pp. 1-55; S. R.Jefferies et al, Journal of Biomedical Materials Research, Vol. 12(1978), pp. 491-503; and U.S. Pat. No. 3,843,446.

Sterile collagen products having a felt or fleece-like structure withopen, communicating voids between the fibers and used as an absorbent inwounds and bone cavities are described in U.S. Pat. No. 4,066,083.

In 1931, Huggins (Arch. Surg., 22:377-408) reported that proliferatingmucosa of Kidney, ureter, or bladder induced bone formation whenimplanted in connective tissue. This was the first reported experimentalmodel of induced ectopic osteogenesis. More recently, Urist (Science,150: 893-899, 1965) and Reddi et al (Proc. Natl. Acad. Sci. U.S.A., 69:1601-1605, 1972) demonstrated that osteogenesis could also be induced bythe devitalized, demineralized matrix of bone or dentin. It has beenshown that physical factors, including surface charge and geometry ofthe matrix, are involved, Reddi et al (Proc. Natl. Acad. Sci. U.S.A.,69: 1601-1605, 1972). There is evidence that a soluble factor fromdemineralized bone, bone morphogenetic protein, is osteo-inductive; seeUrist et al, Proc. Natl. Acad. Sci. U.S.A., 76: 1828-1932, 1979.

In 1899, Senn showed healing of experimental canine calverial defectsand of human tibial and femoral defects with decalcified ovine bone.Others have shown bone formation in periapical areas in dogs and monkeysand in skull defects in rats after implantation or demineralized bone byitself. The osteogenic potential of demineralized bone powder has beendemonstrated in cranial osseous defects in rats. More recently, Mullikenreported on the use of demineralized bone segments, chips, and powderfor reconstruction of craniofacial defents in rats and humans; seeMulliken et al. Plast. Reconstr. Surg., 65: 533-559, 1980 and Glowackiet al, Lancet, May 2, 1981, 963-966.

Histomorphometric evaluation of osteogenesis induced by equal masses ofdemineralized bone powders of various particle sizes, ranging from lessthan 75 millimicrons to greater than 450 millimicrons, has revealed thatsmaller particles induced more bone per field, that is the ratio of bonearea to implant area, than did larger particles. It has also been notedin the literature that large blocks of demineralized cortical boneinduce only a thin layer of new bone on their surfaces. Osteogenesisproceeds more slowly in response to blocks than to powders.

Although blocks, chips, and powders of demineralized bone by itself maybe useful for repair of bony defects, a more defined, better designedmaterial is needed to improve the clinical usefulness of inducedosteogenesis. Greater control is required over the chemical compositionof the graft material than previously accomplished. Banked bone takenfrom cadavers for demineralization (allogenic bone) must be harvestedunder rigid standards and conditions to prevent possible immunologiccomplications or possible transmission of viral or bacterial pathogens.Gamma radiation, one method for sterilization of demineralized bone, mayalter the physio-chemical properties critical for bone induction. It isrecognized that irradiation of demineralized bone powder beforeimplantation weakens the osteogenic response by 20%.

SUMMARY OF THE INVENTION

I have found that complexes of reconstituted collagen with demineralizedbone particles or complexes of reconstituted collagen and solubilizedbone morphogenic protein, optionally with glutaraldehyde as across-linker, with fabricated into sponges or like forms and implantedin vivo in osseous defects induces the formation of osseous tissue inthe animal in which it is implanted. Such complexes are convenientlyfashioned into a suitable form for application/implantation includingthin membranes, gels or preferably in a sponge-like configuration. Thecomplexes of my invention are suitable for many applications includinggrafting implants in plastic and reconstructive surgery, periodontalbone grafting, and in endodontic procedures.

Several properties of demineralized bone and its ability to induceosteogenesis are apparent: particles of demineralized bone appear toinduce greater quantities of new bone than do blocks or chips. Particlesof smaller dimensions induced more bone, expressed as bone area perimplant area, than did larger particles of demineralized bone. Largesections of demineralized bone appear to induce osteogenesis only attheir surface, not deep within the graft itself. Gamma radiation as usedin sterilization appears to decrease osteogenic ability of demineralizedbone material, especially irradiation levels above 1 Mrad of Cobalt 60.A glycoprotein, bone morphogenetic protein (BMP), has been characterizedand is reported to have induced new bone formation in rats. BMP's actiondoes not appear to be species-specific; rabbit BMP has induced new boneformation in rats.

Microparticulate demineralized bone can be complexed with collagendispersions and case into microporous sponges for implantation.Collagen-BMP conjugate sponges can be prepared in a similar fashion.These composite materials have several advantages over banked,demineralized allogenic bone obtained from human cadavers. The novelcomplexes of my invention combine the osteogenic potential ofdemineralized bone with the excellent biocompatibility of reconstitutedcollagen. Collagen, a component of bone matrix, has been shown toincrease fibroblast migration and proliferation; see Ehrmann et al, J.Nat. Can. Inst., 16: 1475-1390 (1956), and to also increase the rate andextent of cell attachment; see also Klebe, Nature, 250: 248-251 (1974).

By the use of demineralized bone powder or bone morphogenic protein as acontrolled spore material to allow cellular ingrowth, osteogenic cellscan proliferate throughout the graft material, allowing bony union ofthe graft and bone formation uniformly within the graft. Greater controlover the chemical composition of the graft material is now possible,thus reducing the possibility of graft-host rejection due tohisto-incompatibility or inflammation. The Collagen-DBP and Collagen-BMPconjugates of my invention increase the available surface area of theimmobilized powder or protein, thus increasing the extent of boneinduction into the implanted mass. Collagen-DBP and Collagen-BMPconjugates confer protection to the physical-chemical properties of thenon-collagen proteins in the conjugate during sterilization by chemicalmeans or irradiation.

Proportions of the BMP and/or DBP may be adjusted within reasonably wideranges depending upon the properties desired and the clinicalapplications required. A majority, i.e., more than 50 weight percent ofthe conjugate material is collagen. Preferably the BMP, DBP or theirmixture are present, in sum, to the extent of from about 5 to about 35weight percent, most preferably from about 10 to about 20 weightpercent, with from about 1 to about 5 weight percent glutaraldehyde,when present, balance collagen. A preferred formulation includes 10 to20 weight percent DBP or BMP, about 1 weight percent glutaraldehyde,balance reconstituted collagen.

The invention will now be further described with reference to thefollowing examples, considered illustrative but not limiting of theinvention. Unless otherwise indicated, all parts and percentages are byweight.

EXAMPLE I A. Preparation of Demineralized Bone Particles

Allogenic bone material was obtained from human cadavers from an organbank. Bones were cleaned and extracted with absolute ethanol followed byanhydrous ethyl ether. The bones were then pulverized in a Spex liquidnitrogen impacting mill and sieved to particle size of less than 75millimicrons (um) to yield bone powder particles.

Demineralized bone powder (DBP) was prepared by extracting thepreviously prepared bone powder particles with 0.5 M HCl (25 Meq/gmbone) for 3 hours at room temperature followed by six washes in steriledistilled water to remove all acids and calcium, followed by foursequential 60-minute washes in absolute ethanol and anhydrous ether.

B. Complexation of DBP with Reconstituted Collagen

Collagen-DBP conjugates were prepared as follows: 10 grams ofpulverized, lyophilized, microcrystalline collagen (Avitene, Avicon,Inc., Fort Worth, Tex.) were dialyzed against sterile distilled waterfor 24 hours to remove the hydrochloric acid salt used in the collagen'spreparation. The collagen was then dispersed in 500 milliliters of 0.05M acetic acid (pH 3.2) by stirring in a refrigerated homogenizer. 1(one) gram of DBP was slowly added to the collagen dispersion. Thisresulted in approximately 10% DBP in the collagen.

Glutaraldehyde cross-linking, an optional but preferred procedure whichincreases the mechanical strength of the conjugate collagen sponge, canbe performed during the homogenization step after the addition of DBP.19.2 milliliters of 25%, biological grade glutaraldehyde (EastmanChemical Co.) were slowly added to the collagen dispersion to create afinal concentration of 1% glutaraldehyde. Alternatively, theglutaraldehyde cross-linking step may be performed after fabrication ofthe lyophilized collagen sponge, in the manner described below.

100 milliliters of the Collagen-DBP dispersion (collagen-particlecomposite material) prepared as described above of approximately 2.2%solids by weight (2% collagen, 0.2% DBP) were then placed in an aluminumcontainer measuring 5 cm×5 cm×5 cm. The container was placed on the -50°C. shelf of a lyophilizer and the contents of the aluminum cube werefrozen thereon. The frozen dispersion was then quickly removed from thecontainer and placed directly in the vacuum chamber of the lyophilizer.The condenser coils were cooled to -150° C. with liquid nitrogen and thevacuum was maintained at approximately 60 mtorr with the use of a pump(Model D150, Precision Scientific, Chicago, Ill.). The specimen wasmaintained at approximately -70° C. during the freeze drying process.The dried specimen was a white, semi-rigid foam.

If the Collagen-DBP conjugate has not been cross-linked earlier in thisprocedure, the sponge can be cross-linked in 1% glutaraldehyde (adjustedto pH 7.0 with sodium phosphate buffer) for 15 minutes at 20° C. undermild stirring. The final concentration of phosphate buffer in thecross-linking bath was 0.01 M. Following cross-linking, the sponge isremoved and placed in a stirred bath of 0.005 M glycine in steriledistilled water for 30 minutes. The sponge is then washed with 10 batchcontacts of sterile distilled water and finally stored in a sealed,sterile container of sterile normal saline.

EXAMPLE II

A procedure described by Urist et al (Proc. Natl. Acad. Sci. U.S.A., 761828-1932, 1979), the disclosure of which is incorporated herein byreference, for isolation of a solubilized bone morphogenetic protein(BMP), which can induce a osteogenesis in vivo, was modified for theisolation of BMP to produce Collagen-BMP bone graft sponges.

Allogenic cortical bone (100 grams) was demineralized in 0.5 M HCl at 4°C. for 24 hours. The demineralized bone was then washed twice in steriledistilled water and then lyophilized. The lyophilized matrix was thensequentially extracted three times with 8 M LiCl to decrease the contentof lipid, proteoglycans, and sialoproteins and to convert the bonecollagen to insoluble bone matrix gelatin.

The bone matrix gelatin was incubated for 24 hours at 37° C. at pH 7.2in a 0.00054% purified bacterial collagenase (Worthington BichemicalCorp.) in Hanks' solution containing 25 mM Tris, 300 mM CaCl₂, 3 mMNaN₂, and adjusted to a pH of 7.2 with 0.1 M HCl. The pH was adjusted to7.2 every two hours for the first eight hours. After 24 hours, the totaldigest was centrifuged at 40,000×G for 15 minutes. The resulting pelletof insoluble collagenase-resistant material was discarded. Thesupernatant, a clear solution, was then dialyzed in cellulose acetatemembrane, 0.30 um pore size, against sterile distilled water for 24hours at 4° C. The dialysate, containing the non-dialyzable substances,was retained and used for complexation with reconstituted collagen asdescribed above.

The complexation of BMP with reconstituted collagen was identical withthe procedure followed in Example I except that BMP solution was addedinstead of DBP particles. Alternatively, one may mix the BMP and DBPsources in complexing with the reconstituted collagen.

EXAMPLE III

An additional modification of the Collagen-DBP and Collagen-BMPconjugates can be accomplished through the additional binding ofauxillary macromolecules in order to modify or accelerate the osteogenicproperties of the conjugate materials. For example, the complexation ofbovine intestinal alkaline phosphatase, at concentrations of 15 mg pergram of Collagen dispersion (either Collagen-DBP of Collagen-BMPdispersions), acts to eliminate all inflammatory responses to the graftmaterial, accelerates the formation of osteoid in the graft material,and slow resorption of the graft enabling it to be more completelycorticalized.

What is claimed is:
 1. A bone graft material capable of inducing theformation of osseous tissue in the animal in which it is implanted, saidbone graft material consisting essentially of a collagen conjugatecontaining:from about 65 to about 95 weight percent reconstitutedcollagen having dispersed substantially uniformly therein from about 35to about 5 weight percent of (a) demineralized bone particles, (b)solubilized bone morphogenic protein, or (c) mixtures of demineralizedbone particles and solubilized bone morphogenic protein, said bone graftmaterial adapted to induce the formation of osseous tissue whenimplanted in an animal.
 2. The bone graft material of claim 1 furtherincluding a cross-linking amount of glutaraldehyde to confer additionalstructural integrity to the bone graft material.
 3. The bone graftmaterial of claim 1 or 2 wherein the demineralized bone powder, whenpresent, has a particle size of no greater than about 70 millimicrons.4. A lyophilized sponge for in vivo implantation composed of the bonegraft material of claim
 1. 5. A lyophilized sponge for in vivoimplantation composed of the bone graft material of claim
 2. 6. Alyophilized sponge for in vivo implantation composed of the bone graftmaterial of claim
 3. 7. The bone graft material of claim 1 or 2 whereinthe amount of reconstituted collagen is about 80 to about 90% and theamount of (a), (b) or (c) is about 20 to about 10%.
 8. The bone graftmaterial of claim 2 wherein the gluteraldehyde is present in an amountof about 1 to about 5 weight percent.
 9. The bone graft material ofclaim 1, 2 or 8 wherein the reconstituted collagen is complexed withalkaline phosphatase.
 10. A biocompatible bone graft material composedof a collagen conjugate and capable of inducing the formation of osseoustissue in the animal in which it is implanted, said bone graft materialconsisting essentially of, in weight percent:(i) from about 65 to about95% reconstituted collagen complexed with alkaline phosphatase, thecollagen having dispersed substantially uniformly therein (ii) fromabout 35% to about 5% of dimineralized bone particles, and (iii) across-linking amount of gluteraldehyde to confer structural integrity tothe bone graft material,said bone graft material adapted to induceosteogenesis, substantially non-inflammatory and biocompatible with thetissue in the animal in which it is implanted.
 11. A biocompatible bonegraft material composed of a collagen conjugate and capable of inducingthe formation of osseous tissue in the animal in which it is implanted,said bone graft material consisting essentially of, in weightpercent:(i) from about 65 to about 95% reconstituted collagen complexedwith alkaline phosphatase, the collagen having dispersed substantiallyuniformly therein (ii) from about 35 to about 5% of solubilized bonemorphogenic protein, and (iii) a cross-linking amount of gluteraldehydeto confer structural integrity to the bone graft material,said bonegraft material adapted to induce osteogenesis, substantiallynon-inflammatory and biocompatible with the tissue in the animal inwhich it is implanted.
 12. The bone graft material of claim 10 or 11wherein the amount of gluteraldehyde is from about 1 to about 5%.